Methods for slowing familial ALS disease progression

ABSTRACT

Methods for slowing disease progression in an individual suffering from familial ALS are provided. Also provided are methods of increasing the survival time of an individual suffering from familial ALS. These methods employ antisense oligonucleotides targeted to SOD1, for use in inhibiting the expression of SOD1 in the central nervous system of an individual suffering from familial ALS.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.11/449,446, filed Jun. 7, 2006, which is a continuation of U.S.application Ser. No 10/672,866, filed Sep. 26, 2003, which is acontinuation-in-part of U.S application Ser. No. 10/633,843, filed Aug.4, 2003, which is a continuation of U.S. application Ser. No.09/888,360, filed Jun. 21, 2001. Further, this application claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser.No. 60/719,936, filed Sep. 21, 2005. The entire contents of thesedocuments are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides compositions and methods for slowingdisease progression in an individual suffering from familial amyotrophiclateral sclerosis. In particular, this invention relates to antisensecompounds, particularly antisense oligonucleotides, complementary toSOD1 nucleic acids. Such antisense oligonucleotides have been shown toinhibit the expression of SOD1.

BACKGROUND OF THE INVENTION

The superoxide anion (O₂ ⁻) is a potentially harmful cellular by-productproduced primarily by errors of oxidative phosphorylation inmitochondria (Cleveland and Liu, Nat. Med., 2000, 6, 1320-1321). Some ofthe targets for oxidation by superoxide in biological systems includethe iron-sulfur dehydratases, aconitase and fumarases. Release of Fe(II) from these superoxide-inactivated enzymes results in Fenton-typeproduction of hydroxyl radicals which are capable of attacking virtuallyany cellular target, most notably DNA (Fridovich, Annu. Rev. Biochem.,1995, 64, 97-112).

The enzymes known as the superoxide dismutases (SODs) provide defenseagainst oxidative damage of biomolecules by catalyzing the dismutationof superoxide to hydrogen peroxide (H₂O₂) (Fridovich, Annu. Rev.Biochem., 1995, 64, 97-112). Two major classes of superoxide dismutasesexist. One consists of a group of enzymes with active sites containingcopper and zinc while the other class has either manganese or iron atthe active site (Fridovich, Annu. Rev. Biochem., 1995, 64, 97-112).

The soluble superoxide dismutase 1 enzyme (also known as SOD1 and Cu/Znsuperoxide dismutase) contains a zinc- and copper-type active site(Fridovich, Annu. Rev. Biochem., 1995, 64, 97-112). Lee et al. reportedthe molecular cloning and high-level expression of human cytoplasmicsuperoxide dismutase gene in E. coli in 1990 (Lee et al., MisaengmulHakhoechi, 1990, 28, 91-97). Studies of transgenic mice carrying amutant human superoxide dismutase 1 gene, for example, transgenic miceexpressing a human SOD1 gene bearing glycine 93 to alanine (G93A)mutation.

Mutations in the superoxide dismutase 1 gene are associated with adominantly-inherited form of amyotrophic lateral sclerosis (ALS, alsoknown as Lou Gehrig's disease) a disorder characterized by a selectivedegeneration of upper and lower motor neurons (Cleveland and Liu, Nat.Med., 2000, 6, 1320-1321). The deleterious effects of various mutationson superoxide dismutase 1 are most likely mediated through a gain oftoxic function rather than a loss of superoxide dismutase 1 activity, asthe complete absence of superoxide dismutase 1 in mice neitherdiminishes life nor provokes overt disease (Al-Chalabi and Leigh, Curr.Opin. Neurol., 2000, 13, 397-405; Alisky and Davidson, Hum. Gene Ther.,2000, 11, 2315-2329).

Cleveland and Liu proposed two models for mutant superoxide dismutase 1toxicity (Cleveland and Liu, Nat. Med., 2000, 6, 1320-1321). The“oxidative hypothesis” ascribes toxicity to binding of aberrantsubstrates such as peroxynitrite or hydrogen peroxide which gain accessto the catalytic copper ion through mutation-dependent loosening of thenative superoxide dismutase 1 protein conformation (Cleveland and Liu,Nat. Med., 2000, 6, 1320-1321). A second possible mechanism for mutantsuperoxide dismutase 1 toxicity involves the misfolding and aggregationof mutant superoxide dismutase 1 proteins (Cleveland and Liu, Nat. Med.,2000, 6, 1320-1321). The idea that aggregates contribute to ALS hasreceived major support from the observation that murine models ofsuperoxide dismutase 1 mutant-mediated disease feature prominentintracellular inclusions in motor neurons and, in some cases, in theastrocytes surrounding them as well (Bruijn et al., Science, 1998, 281,1851-1854). Furthermore, Bruijn et al. also demonstrate that neitherelimination nor elevation of wild-type superoxide dismutase 1 was foundto affect disease induced by mutant superoxide dismutase 1 in mice(Bruijn et al., Science, 1998, 281, 1851-1854).

Riluzole, a glutamate regulatory drug, is approved for use in ALSpatients in some countries, but has only a modest effect on survival.Accordingly, there remains an unmet need for therapeutic regimens thatslow familial ALS disease progression and increase survival of familialALS patients.

SUMMARY OF THE INVENTION

The present invention provides methods of slowing disease progression inan individual suffering from familial amyotrophic lateral sclerosis(ALS), comprising administering to the individual a therapeuticallyeffective amount of a pharmaceutical composition comprising an antisenseoligonucleotide 17 to 25 nucleobases in length complementary tonucleobases 66 to 102 of SEQ ID NO: 1, thereby slowing diseaseprogression. The administering may comprise delivery to thecerebrospinal fluid of the individual, and may further compriseintrathecal infusion. A slowing of disease progression is measured by animprovement in one or more indicators of ALS disease progressionselected from ALSFRS-R, FEV₁, FVC, or muscle strength measurements. Themethods further comprise increasing the survival time of an individualsuffering from familial ALS.

The methods provided herein comprise the administration of an antisenseoligonucleotide complementary to nucleobases 83 to 102 of SEQ ID NO: 1.The antisense oligonucleotide may be fully complementary to nucleotides83 to 102 of SEQ ID NO: 1. Further, the antisense oligonucleotide mayconsist essentially of ISIS 333611. Additionally, the antisenseoligonucleotide may consist of ISIS 333611.

The antisense oligonucleotides employed in the methods provided hereincomprise at least one modified internucleoside linkage, such as aphosphorothioate internucleoside linkage, a modified sugar moiety, suchas a 2′-O-methoxyethyl sugar moiety or a bicyclic nucleic acid sugarmoiety, or a modified nucleobase, such as a 5-methylcytosine.

The present invention provides methods of slowing disease progression inan individual suffering from familial ALS comprising administering anantisense oligonucleotide that is a chimeric oligonucleotide. Thechimeric oligonucleotide comprises a 2′-deoxynucleotide gap segmentpositioned between 5′ and 3′ wing segments. The wing segments arecomprised of nucleosides containing a sugar moiety selected from a2′-O-methoxyethyl sugar moiety or a bicyclic nucleic acid sugar moiety.The gap segment may be ten 2′-deoxynucleotides in length and each of thewing segments may be five 2′-O-methoxyethyl nucleotides in length. Thechimeric oligonucleotide may be uniformly comprised of phosphorthioateinternucleoside linkages. Further, each cytosine of the chimericoligonucleotide may be a 5′-methylcytosine.

Further provided are methods comprising selecting an individual who hasreceived a diagnosis of familial amyotrophic lateral sclerosis (ALS),administering to the individual a therapeutically effective amount of apharmaceutical composition comprising an antisense oligonucleotide 17 to25 nucleobases in length complementary to nucleobases 66 to 102 of SEQID NO: 1, and monitoring ALS disease progression in the individual.

DETAILED DESCRIPTION OF THE INVENTION

Over 100 mutations of the human SOD1 gene have been identified, andaltogether account for approximately 20% of familial amyotrophic lateralsclerosis (ALS) cases. Some mutations, such as the A4V mutation mostcommonly found in the United States, are highly lethal and result insurvival only nine months from the onset of disease symptoms. Othermutations of SOD1 manifest in a slower disease course.

It has been discovered that antisense inhibition of superoxide dismutase1 (SOD1) in an animal model of familial ALS reduces both SOD1 mRNA andprotein, and further results in a slowing of disease progression and,importantly, increased survival time. Accordingly, the present inventionprovides methods for the slowing of disease progression in an individualsuffering from familial ALS by delivering to the cerebrospinal fluid ofthe individual a therapeutically effective amount of a pharmaceuticalcomposition comprising an antisense oligonucleotide targeted to SOD1.Such methods further comprise increasing survival time of an individualsuffering from familial ALS. Slowing of disease progression is indicatedby an improvement in one or more indicators of ALS disease progression,including, without limitation, the revised ALS functional rating scale,pulmonary function tests, and muscle strength measurements.

The present invention employs antisense compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding SOD1, ultimately modulating the amountof SOD1 protein produced. This is accomplished by providing antisenseoligonucleotides which hybridize to and inhibit the expression of one ormore nucleic acids encoding SOD1. Such antisense oligonucleotides areconsidered to be “targeted to SOD1.” Antisense oligonucleotides of thepresent invention do not necessarily distinguish between wild-type SOD1mRNA and SOD1 mRNAs bearing mutations. While an object of the presentinvention is to reduce SOD1 mRNAs bearing mutations, the conconmitantreduction of wild-type SOD1 appear to be safe, as reductions or completeloss of SOD1 does not produce overt disease or compromise life span inexperimental animal models.

In certain embodiments, an antisense oligonucleotide targeted to SOD1 iscomplementary to nucleobases 66 to 102 of a nucleic acid moleculeencoding SOD1 (GENBANK® accession no. X02317.1, incorporated herein asSEQ ID NO: 1). In additional embodiments, an antisense oligonucleotidetargeted to SOD1 is complementary to nucleotides 83 to 102 of SEQ IDNO: 1. In preferred embodiments, an antisense oligonucleotide targetedto SOD1 is fully complementary to nucleotides 83 to 102 of SEQ ID NO: 1.In further preferred embodiments, the antisense oligonucleotide is ISIS333611.

As used herein, an “individual suffering from familial ALS” is anindividual who has received from a health professional, such as aphysician, a diagnosis of familial ALS. Relevant diagnostic tests arewell known in the art and are understood to include, without limitation,genetic testing to determine the presence of a mutation in the SOD1gene, neurological examination, and the El Escorial criteria (see, forexample, Brooks et al., Amyothoph. Lateral Scler. other Motor NeuronDisorders, 2000, 293-299). An “individual prone to familial ALS” isunderstood to include an individual who, based on a physician'sassessment, is not yet suffering from familial ALS but is likely todevelop familial ALS.

In order for antisense inhibition of SOD1 to have a clinically desirableeffect on familial ALS progression, it is beneficial to deliver anantisense oligonucleotide targeted to SOD1 to the central nervous system(CNS) of an individual suffering from familial ALS, and in particular tothe regions of the CNS affected by familial ALS. As the blood-brainbarrier is generally impermeable to antisense oligonucleotidesadministered systemically, a preferred method of providing antisenseoligonucleotides targeted to SOD1 to the tissues of the CNS is viaadministration of the antisense oligonucleotides directly into thecerebrospinal fluid (CSF). As is known in the art, means for delivery tothe CSF include intrathecal (IT) and intracerebroventricular (ICV)administration. As is further known in the art, IT or ICV administrationmay be achieved through the use of surgically implanted pumps thatinfuse a therapeutic agent into the cerebrospinal fluid. As used herein,“delivery to the CSF” and “administration to the CSF” encompass the ITinfusion or ICV infusion of antisense oligonucleotides targeted to SOD1through the use of an infusion pump. In some embodiments, IT infusion isa preferred means for delivery to the CSF. In preferred embodiments, theantisense oligonucleotide is continuously infused into the CSF for theentire course of treatment; such administration is referred to as“continuous infusion” or, in the case of IT infusion, “continuous ITinfusion.”

In the context of the present invention, a preferred means for deliveryof antisense oligonucleotide to the CSF employs an infusion pump such asMedtronic SyncroMed® II pump. The SyncroMed® II pump is surgicallyimplanted according the procedures set forth by the manufacturer. Thepump contains a reservoir for retaining a drug solution, which is pumpedat a programmed dose into a catheter that is surgically implanted. Forintrathecal administration of a drug, the catheter is surgicallyintrathecally implanted. In the context of the present invention, thedrug is the pharmaceutical composition comprising an antisenseoligonucleotide targeted to SOD1.

As used herein, a “pharmaceutical composition comprising an antisenseoligonucleotide” refers to a composition comprising an antisenseoligonucleotide targeted to SOD1 in a pharmaceutically acceptablediluent. By way of example, a suitable pharmaceutically acceptablediluent is phosphate-buffered saline. In some embodiments, thepharmaceutical composition comprises an antisense oligonucleotidecomplementary to nucleotides 66 to 102 of SOD1 in phosphate-bufferedsaline. In preferred embodiments, the pharmaceutical compositioncomprises an antisense oligonucleotide complementary to nucleotides 83to 102 of SEQ ID NO: 1 in phosphate-buffered saline. In furtherpreferred embodiments, the pharmaceutical composition comprises anantisense oligonucleotide fully complementary to nucleotides 83 to 102of SEQ ID NO: 1 in phosphate-buffered saline. In more preferredembodiments, the pharmaceutical composition comprises ISIS 333611 inphosphate-buffered saline. ISIS 333611 is the nonadecasodium salt of theantisense oligonucleotide having the nucleobase sequenceCCGTCGCCCTTCAGCACGCA (SEQ ID NO: 2), where nucleosides 1 to 5 and 16 to20 have 2′-O-methoxyethyl sugar moieties, nucleosides 6 to 15 are2′-deoxynucleotides, each internucleoside linkage is a phosphorothioatelinkage, and each cytosine is a 5-methylcytosine.

As used herein, a “therapeutically effective amount” is an amount of anantisense oligonucleotide targeted to SOD1 required to produce a slowingof disease progression and/or an increase in survival time in anindividual suffering from familial ALS. Accordingly, a therapeuticallyeffect amount is an amount that will result in an improvement in one ormore indicators of ALS progression, such as, for example, the revisedALSFSR, FEV₁, FCV, and muscle strength measurements. In someembodiments, a therapeutically effective amount of an antisenseoligonucleotide targeted to SOD1 ranges from 8 mg to 12 mg of antisenseoligonucleotide. In preferred embodiments, a therapeutically effectamount of an antisense oligonucleotide targeted to SOD1 is 10 mg. In oneembodiment, a therapeutically effective amount of an antisenseoligonucleotide targeted to SOD1 is administered via continuous infusionfor a minimum of 28 days. In preferred embodiments, antisenseoligonucleotide is delivered via IT infusion. In further preferredembodiments, the antisense oligonucleotide administered is ISIS 333611.

As used herein, “slowing disease progression” means the prevention of aclinically undesirable change in one or more disabilities in anindividual suffering from familial ALS, and is assessed by methodsroutinely practiced in the art, for example, the revised ALSFSR,pulmonary function tests, and muscle strength measurements. Such methodsare herein referred to as “indicators of ALS disease progression.”

As used herein, an “improvement in a indicator of ALS diseaseprogression” refers to slowing of the rate of change in one or more ofthe indicators of ALS disease progression described herein. Animprovement in an indicator of ALS disease progression further includesa lack of a measurable change in one or more of the indicators of ALSdisease progression described herein. An improvement in an indicator ofALS disease progression additionally includes a positive change in oneof the indicators of ALS disease progression described herein, such as,for example, an increase in an ALSFSR-R score. One of skill in the artwill appreciate that is well within the abilities of a physician toidentify a slowing of disease progression in an individual sufferingfrom familial ALS, using one or more of the disease assessment testsdescribed herein. Additionally, it is understood that a physician mayadminister to the individual diagnostic tests other than those describedherein, such as additional pulmonary function tests or muscle strengthmeasurement tests, to assess the rate of disease progression in anindividual suffering from familial ALS.

A slowing of disease progression may further comprise an “increase insurvival time” in an individual suffering from familial ALS. It isunderstood that a physician can use one or more of the diseaseassessment tests described herein to predict an approximate survivaltime of an individual suffering from familial ALS. A physician mayadditionally use the known disease course of a particular familial ALSmutation to predict survival time.

The “revised ALS functional rating scale” or “ALSFRS-R” is routinelyused by physicians and is a validated rating instrument for monitoringthe progression of disability in ALS patients. The ALSFRS-R includes 12questions that ask a physician to rate his or her impression of an ALSpatient's level of functional impairment in performing one of ten commontasks, for example, climbing stairs. Each task is rated on a five-pointscale, where a score of zero indicates an inability to perform a taskand a score of four indicates normal ability in performing a task.Individual item scores are summed to produce a reported score of betweenzero (worst) and 48 (best).

ALS eventually results in progressive muscle weakness. Pulmonaryfunction tests (PFTs) are employed to reveal the extent and progressionrespiratory muscle weakness. Pulmonary function tests includemeasurements of the “forced expiratory volume in one second” (FEV₁),which is the amount of air than an individual can forcefully exhaleduring the first second of exhalation following inhalation, as well asmeasurements of “forced vital capacity” (FVC), which is the total amountof air that an individual can forcefully exhale following inhalation.FEV₁ and FVC are typically reduced in individuals with neuromusculardiseases, such as ALS. Pulmonary function tests may be administeredusing instrument that directly measures the volume of air displacedduring exhalation, or measures airflow during exhalation by a flowsensing device. An example of such an instrument is a spirometer. ALSdisease progression is also evaluated by assessing the extent andprogression of whole body muscle weakness. Muscle strength measurements,include, but are not limited to, hand held dynamometry, maximumvoluntary isometric contraction (MVIC) strain gauge measurements, andmanual muscle testing. Muscle strength tests routinely use an instrumentthat measures how much force (for example, pounds of force) anindividual can apply to the instrument using a selected group ofmuscles, such as the hand muscles. Such an instrument includes adynamometer.

Antisense Compounds

In the context of the present invention, the term “oligomericcompound(s)” refers to polymeric structures which are capable ofhybridizing to at least a region of an RNA molecule. Generally, anoligomeric compound is “antisense” to a target nucleic acid when itcomprises the reverse complement of the corresponding region of thetarget nucleic acid. Such oligomeric compounds are known as “antisensecompounds”, which include, without limitation, oligonucleotides (i.e.antisense oligonucleotides), oligonucleosides, oligonucleotide analogs,oligonucleotide mimetics and combinations of these. In general, anantisense compound comprises a backbone of linked monomeric subunits(sugar moieties) where each linked monomeric subunit is directly orindirectly attached to a heterocyclic base moiety. Modifications toantisense compounds encompass substitutions or changes tointernucleoside linkages, sugar moieties, or heterocyclic base moieties,such as those described below. As used herein, the term “modification”includes substitution and/or any change from a starting or naturalnucleoside or nucleotide. Modified antisense compounds are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for nucleic acidtarget, increased stability in the presence of nucleases, or increasedinhibitory activity. Antisense compounds are often defined in the art tocomprise the further limitation of, once hybridized to a target, beingable to induce or trigger a reduction in target gene expression ortarget gene levels. In one embodiment, the antisense compounds, e.g.antisense oligonucleotides, trigger a reduction in the levels of anucleic acid encoding SOD1.

“Targeting” an antisense oligonucleotide to a nucleic acid encoding SOD1includes the determination of at least one target segment within anucleic acid encoding SOD1 for hybridization to occur such that thedesired effect, e.g., inhibition of SOD1 mRNA expression, will result.As used herein, the terms “SOD1 target nucleic acid” and “nucleic acidencoding SOD1” encompass RNA (including pre-mRNA and mRNA) transcribedfrom DNA encoding SOD1, and also cDNA derived from such RNA. Theinhibition of gene expression that results from the hybridization of anantisense oligonucleotide with a target nucleic acid is generallyreferred to as “antisense inhibition”. The functions of RNA to beinterfered with include, for example, translocation of the RNA to thesite of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in or facilitated by the RNA. The overalleffect of such interference with target nucleic acid function ismodulation of the SOD1 protein. In the context of the present invention,“modulation” means either an increase (stimulation) or a decrease(inhibition) in the expression of a gene. In the context of the presentinvention, inhibition is the preferred form of modulation of geneexpression and SOD1 mRNA (e.g. SEQ ID NO: 1) is a preferred target.

As used herein, a “target segment” means a sequence of an SOD1 targetnucleic acid to which one or more antisense oligonucleotides arecomplementary. Multiple antisense oligonucleotides complementary to agiven target segment may or may not have overlapping sequences. Withinthe context of the present invention, the term “target site” is definedas a sequence of an SOD1 nucleic acid to which one antisenseoligonucleotide is complementary. For example, the nucleobase sequenceof ISIS 333611 is complementary to nucleobases 83 to 102 of SEQ ID NO:1, thus these nucleobases represent a target site of an SOD1 nucleicacid. Several antisense oligonucleotides of the invention have targetsites within nucleobases 66 to 102 of SEQ ID NO: 1, thus thesenucleobases represent a target segment of an SOD1 nucleic acid. In someembodiments, a target segment and target site are represented by thesame nucleobase sequence.

In the practice of the methods of the present invention, particularlypreferred SOD1 target segments include, without limitation, nucleobases66 to 102 of SEQ ID NO: 1; nucleobases 73 to 102 of SEQ ID NO: 1; andnucleobases 79 to 102 of SEQ ID NO: 1. Particularly preferred targetsites include nucleobases 81 to 100 of SEQ ID NO: 1, to which ISIS146145 (SEQ ID NO. 3) is complementary; and nucleobases 83 to 102 of SEQID NO: 1, to which ISIS 333611 (SEQ ID NO. 2) is complementary.

The antisense oligonucleotides in accordance with this inventioncomprise from 15 to 30 to nucleosides in length, i.e., from 15 to 30linked nucleosides. One of skill in the art will appreciate that thisembodies antisense oligonucleotides of 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 nucleosides in length.

In one embodiment, the antisense oligonucleotides of the invention are17 to 25 nucleosides in length, as exemplified herein.

In preferred embodiments, the antisense oligonucleotides of theinvention are 19, 20, 21, 22 or 23 nucleosides in length.

“Base complementarity” as used herein, refers to the capacity for theprecise base pairing of nucleobases of an antisense oligonucleotide withcorresponding nucleobases in a target nucleic acid (i.e.,hybridization). In the context of the present invention, the mechanismof pairing involves hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between correspondingnucleobases. Both natural and modified nucleobases are capable ofparticipating in hydrogen bonding. Hybridization can occur under varyingcircumstances.

As used herein, an antisense oligonucleotide is “fully complementary” toa target nucleic acid when each nucleobase of the antisenseoligonucleotide is capable of undergoing precise base pairing with anequal number of nucleobases in the target nucleic acid. It is understoodin the art that the sequence of the antisense oligonucleotide need notbe fully complementary to that of its target nucleic acid to be activein inhibiting the activity of the target nucleic acid. In someembodiments there are “non-complementary” positions, also known as“mismatches”, between the antisense oligonucleotide and the targetnucleic acid, and such non-complementary positions may be toleratedbetween an antisense oligonucleotide and the target nucleic acidprovided that the antisense oligonucleotide remains specificallyhybridizable to the target nucleic acid. For example, ISIS 333611,having two non-complementary nucleobases with respect to monkey SOD1, iscapable of reducing monkey SOD1 mRNA levels in cultured cells. A“non-complementary nucleobase” means a nucleobase of an antisenseoligonucleotide that is unable to undergo precise base pairing with anucleobase at a corresponding position in a target nucleic acid. As usedherein, the terms “non-complementary” and “mismatch” are interchangable.In the context of the present invention, antisense oligonucleotideshaving no more than three non-complementary nucleobases with respect toa nucleic acid encoding SOD1 are considered “complementary” to a nucleicacid encoding SOD1. In preferred embodiments, the antisenseoligonucleotide contains no more than two non-complementary nucleobaseswith respect to a nucleic acid encoding SOD1. In further preferredembodiments, the antisense oligonucleotide contains no more than onenon-complementary nucleobases with respect to a nucleic acid encodingSOD1.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the antisense oligonucleotide. Alternatively, thenon-complementary nucleobase may be at an internal position in theantisense oligonucleotide. When two or more non-complementarynucleobases are present, they may be contiguous (i.e. linked) ornon-contiguous.

In other embodiments of the invention, the antisense oligonucleotidescomprise at least 90% sequence complementarity to an SOD1 target nucleicacid. In further embodiments of the invention, the antisenseoligonucleotides comprise at least 95% sequence complementarity to anSOD1 target nucleic acid. Percent complementarity of an antisenseoligonucleotide with a region of a target nucleic acid can be determinedroutinely by those having ordinary skill in the art, and may beaccomplished using BLAST programs (basic local alignment search tools)and PowerBLAST programs known in the art (Altschul et al., J. Mol.Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656).

Antisense oligonucleotides may have a defined percent identity to a SEQID NO, or an antisense oligonucleotide having a specific ISIS number.This identity may be over the entire length of the antisenseoligonucleotide, or over less than the entire length of the antisenseoligonucleotide. Calculating percent identity is well within the abilityof those skilled in the art. It is understood by those skilled in theart that an antisense oligonucleotide need not have an identicalsequence to those described herein to function similarly to theantisense oligonucleotides described herein. For example, antisenseoligonucleotides having at least 90%, or at least 95%, identity toantisense oligonucleotides taught herein are contemplated in the presentinvention.

Shortened or truncated versions of antisense oligonucleotides taughtherein have one, two or more nucleosides deleted, and are contemplatedin the present invention. When an antisense oligonucleotide has two ormore deleted nucleosides, the deleted nucleosides may be adjacent toeach other, for example, in an antisense oligonucleotide having twonucleosides truncated from the 5′ end (5′ truncation), or alternativelyfrom the 3′ end (3′ truncation), of the antisense oligonucleotide.Alternatively, the deleted nucleosides may be dispersed through out theantisense, for example, in an antisense oligonucleotide having onenucleoside deleted from the 5′ end and one nucleoside deleted from the3′ end.

Also falling within the scope of the invention are lengthened versionsof antisense oligonucleotides taught herein, i.e. antisenseoligonucleotides having one or more additional nucleosides relative toan antisense oligonucleotide disclosed herein. When two are moreadditional nucleosides are present, the added nucleosides may beadjacent to each other, for example, in an antisense oligonucleotidehaving two nucleosides added to the 5′ end (5′ addition), oralternatively to the 3′ end (3′ addition), of the antisenseoligonucleotide. Alternatively, the added nucleosides may be dispersedthroughout the antisense oligonucleotide, for example, in an antisenseoligonucleotide having one nucleoside added to the 5′ end and onenucleoside added to the 3′ end.

Antisense oligonucleotides of the invention may be also be described ascomplementary to a portion of a target site. A “portion” is defined asat least 18 contiguous nucleobases of a target site. In otherembodiments, a portion is 19 or 20 contiguous nucleobases of a targetsite. By way of example, antisense oligonucleotides may becomplementary, or alternatively fully complementary, to a 19 nucleobaseportion of SEQ ID NO: 1.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules.

Antisense Compound Modifications

Any of the antisense compounds taught herein, including antisenseoligonucleotides taught herein, may contain modifications which conferdesirable properties to the antisense compound including, but are notlimited to, increased affinity of an antisense oligonucleotide for itstarget RNA and increased resistance to nucleases.

As is known in the art, a nucleoside is a base-sugar combination. Thebase (also known as nucleobase) portion of the nucleoside is normally aheterocyclic base moiety. Nucleotides are nucleosides that furtherinclude a phosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety ofthe sugar. In forming oligonucleotides, the phosphate groups covalentlylink adjacent nucleosides to one another to form a linear polymericcompound. The respective ends of this linear polymeric structure can bejoined to form a circular structure by hybridization or by formation ofa covalent bond. Within the oligonucleotide structure, the phosphategroups are commonly referred to as forming the internucleoside linkagesof the oligonucleotide. The normal internucleoside linkage of RNA andDNA is a 3′ to 5′ phosphodiester linkage.

In the context of this invention, the term “oligonucleotide” refersgenerally to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA), and may be used to refer to unmodifiedoligonucleotides or oligonucleotide analogs. The term “unmodifiedoligonucleotide” refers generally to oligonucleotides composed ofnaturally occuring nucleobases, sugars, and covalent internucleosidelinkages. The term “oligonucleotide analog” refers to oligonucleotidesthat have one or more non-naturally occurring nucleobases, sugars,and/or internucleoside linkages. Such non-naturally occurringoligonucleotides are often selected over naturally occurring formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for target nucleic acids, increased stabilityin the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase thebinding affinity of a shortened or truncated antisense compound for itstarget nucleic acid. Consequently, comparable results can often beobtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

Specific examples of antisense compounds useful in this inventioninclude oligonucleotides containing one or more modified, i.e.non-naturally occurring, internucleoside linkages. Such non-naturallyinternucleoside linkages are often selected over naturally occurringforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for other oligonucleotides or nucleicacid targets and increased stability in the presence of nucleases.

As defined in this specification, oligonucleotides having modifiedinternucleoside linkages include internucleoside linkages that retain aphosphorus atom as well as internucleoside linkages that do not have aphosphorus atom. Representative phosphorus containing internucleosidelinkages include, but are not limited to, phosphodiesters,phosphotriesters, methylphosphonates, phosphoramidate, andphosphorothioates. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be “oligonucleosides”. Methods of preparation ofphosphorous-containing and non-phosphorous-containing linkages are wellknown to those skilled in the art.

Modified Sugar Moieties

Antisense compounds of the invention may also contain one or morenucleosides having modified sugar moieties. The base moieties (natural,modified or a combination thereof) are maintained for hybridization withan appropriate nucleic acid target. Sugar modifications may impartnuclease stability, binding affinity or some other beneficial biologicalproperty to the antisense compounds. The furanosyl sugar ring of anucleoside can be modified in a number of ways including, but notlimited to, addition of a substituent group, particularly at the 2′position, bridging of two non-geminal ring atoms to form a bicyclicnucleic acid (BNA) and substitution of an atom or group such as —S—,—N(R)— or —C(R₁)(R₂) for the ring oxygen at the 4′-position. Arepresentative list of preferred modified sugars includes but is notlimited to: substituted sugars, especially 2′-substituted sugars havinga 2′-F, 2′-OCH₂ (2′-OMe) or a 2′-O(CH₂)₂—OCH₃ (2′-O-methoxyethyl or2′-MOE) substituent group; and bicyclic modified sugars (BNAs), having a4′-(CH₂)_(n)—O-2′ bridge, where n=1 or n=2. Sugars can also be replacedwith sugar mimetic groups. Methods for the preparations of modifiedsugars are well known to those skilled in the art.

Modified Nucleobases

Antisense compounds of the invention may also contain one or morenucleobase (often referred to in the art simply as “base”) modificationsor substitutions which are structurally distinguishable from, yetfunctionally interchangeable with, naturally occurring or syntheticunmodified nucleobases. Such nucleobase modifications may impartnuclease stability, binding affinity or some other beneficial biologicalproperty to the antisense compounds. As used herein, “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases also referred to herein as heterocyclic basemoieties include other synthetic and natural nucleobases such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and otheralkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases that are particularly useful for increasing the bindingaffinity of the antisense compounds of the invention includ5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2 aminopropyladenine, 5-propynyluraciland 5-propynylcytosine.

Certain nucleobase substitutions, including 5-methylcytosinsesubstitutions, are particularly useful for increasing the bindingaffinity of the oligonucleotides of the invention. For example,5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Oligonucleotide Mimetics

Antisense compounds can also include an “oligonucleotide mimetic,” whichrefers to oligonucleotides in which only the furanose ring or both thefuranose ring and the internucleoside linkage are replaced with novelgroups.

As used herein the term “mimetic” refers to groups that are substitutedfor a sugar, a nucleobase, and/or internucleoside linkage. Generally, amimetic is used in place of the sugar or sugar-internucleoside linkagecombination, and the nucleobase is maintained for hybridization to aselected target. Representative examples of a sugar mimetic include, butare not limited to, cyclohexenyl or morpholino. Representative examplesof a mimetic for a sugar-internucleoside linkage combination include,but are not limited to, peptide nucleic acids (PNA) and morpholinogroups linked by uncharged achiral linkages. In some instances a mimeticis used in place of the nucleobase. Representative nucleobase mimeticsare well known in the art and include, but are not limited to, tricyclicphenoxazine analogs and universal bases (Berger et al., Nuc. Acid Res.2000, 28:2911-14, incorporated herein by reference). Methods ofsynthesis of sugar, nucleoside and nucleobase mimetics are well known tothose skilled in the art.

Conjugated Antisense Compounds

One substitution that can be appended to the antisense compounds of theinvention involves the linkage of one or more moieties or conjugateswhich enhance the activity, cellular distribution or cellular uptake ofthe resulting antisense compounds. In one embodiment such modifiedantisense compounds are prepared by covalently attaching conjugategroups to functional groups such as hydroxyl or amino groups. Conjugategroups of the invention include intercalators, reporter molecules,polyamines, polyamides, polyethylene glycols, polyethers, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of the antisense compounds.Typical conjugate groups include cholesterol moieties and lipidmoieties. Additional conjugate groups include carbohydrates,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds used in the compositions of the present inventioncan also be modified to have one or more stabilizing groups that aregenerally attached to one or both termini of antisense compounds toenhance properties such as, for example, nuclease stability. Included instabilizing groups are cap structures. By “cap structure” or “terminalcap moiety” is meant chemical modifications, which have beenincorporated at either terminus of an antisense compound (see forexample Wincott et al., WO 97/26270). These terminal modificationsprotect the antisense compounds having terminal nucleic acid moleculesfrom exonuclease degradation, and can help in delivery and/orlocalization within a cell. The cap can be present at the 5′-terminus(5′-cap), or at the 3′-terminus (3′-cap), or can be present on bothtermini. For double-stranded antisense compounds, the cap may be presentat either or both termini of either strand. Cap structures are wellknown in the art and include, for example, inverted deoxy abasic caps.Further 3′ and 5′-stabilizing groups that can be used to cap one or bothends of an antisense compound to impart nuclease stability include thosedisclosed in WO 03/004602 published on Jan. 16, 2003.

Antisense Compound Motifs

Antisense compounds of this invention may have the chemically modifiedsubunits arranged in patterns enhance the inhibitory activity of theantisense compounds. These patterns are described herein as “motifs.”

As used in the present invention the term “gapped motif” or “gapmer” ismeant to include an antisense compound having an internal region (alsoreferred to as a “gap” or “gap segment”) positioned between two externalregions (also referred to as “wing” or “wing segment”). The regions aredifferentiated by the types of sugar moieties comprising each distinctregion. The types of sugar moieties that are used to differentiate theregions of a gapmer may in some embodiments include β-D-ribonucleosides,β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modifiednucleosides may include 2′-MOE, and 2′-O—CH₃, among others), andbicyclic sugar modified nucleosides (such bicyclic sugar modifiednucleosides may include LNA™ or ENA™, among others). In general, eachdistinct region comprises uniform sugar moieties.

Gapped motifs or gapmers are further defined as being either “symmetric”or “asymmetric”. A gapmer wherein the nucleosides of the first wing havethe same sugar modifications as the nucleosides of the second wing istermed a symmetric gapped antisense compound. Symmetric gapmers canhave, for example, an internal region comprising a first sugar moiety,and external regions each comprising a second sugar moiety, wherein atleast one sugar moiety is a modified sugar moiety.

“Chimeric antisense compounds” or “chimeras,” in the context of thisinvention, are antisense compounds that at least 2 chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotideor nucleoside in the case of a nucleic acid based antisense compound.Accordingly, antisense compounds having a gapmer motif consideredchimeric antisense compounds.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. By way of example,an antisense compound may be designed to comprise a region that servesas a substrate for the cellular endonuclease RNase H, which cleaves theRNA strand of an RNA:DNA duplex. In the case of gapmer, the internalregion generally serves as the substrate for endonuclease cleavage.

Compositions and Methods for Formulating Pharmaceutical Compositions

The antisense compounds of the invention may also be admixed withpharmaceutically acceptable substances, active and/or inert, that arewell known to those skilled in the art.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds and compositions ofthe invention. Compositions and methods for the formulation ofpharmaceutical compositions are dependent upon a number of criteria,including, but not limited to, route of administration, extent ofdisease, or dose to be administered. Such considerations are wellunderstood by those skilled in the art.

The antisense compounds and compositions of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofthe compound or composition to a suitable pharmaceutically acceptablediluent or carrier. In the context of the present invention, apharmaceutically acceptable diluent includes phosphate-buffered saline(PBS). PBS is a diluent suitable for use in compositions to be deliveredto the CNS.

The antisense compounds and compositions of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the antisense compounds of the invention, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. This can include the incorporation ofadditional nucleosides at one or both ends of an antisense compoundwhich are cleaved by endogenous nucleases within the body, to form theactive antisense compound.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the antisense compounds andcompositions of the invention: i.e., salts that retain the desiredbiological activity of the parent compound and do not impart undesiredtoxicological effects thereto. Suitable examples include, but are notlimited to, sodium and potassium salts.

Cell Culture and Antisense Oligonucleotide Treatment

The effects of antisense oligonucleotides on the level, activity orexpression of SOD1 target nucleic acids can be tested in vitro in avariety of cell types. Cell types used for such analyses are availablefrom commerical vendors (e.g. American Type Culture Collection,Manassus, Va.; Zen-Bio, Inc., Research Triangle Park, NC; CloneticsCorporation, Walkersville, Md.) and cells are cultured according to thevendor's instructions using commercially available reagents (e.g.Invitrogen Life Technologies, Carlsbad, Calif.). Illustrative cell typesinclude, but are not limited to, A549 cells, fibroblasts, and neuronalcells.

In vitro Testing of Antisense Oligonucleotides

In general, when cells reach approximately 60-80% confluency, they aretreated with antisense oligonucleotides of the invention.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE® (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with LIPOFECTAMINE® in OPTI-MEM® 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a LIPOFECTAMINE®concentration that typically ranges 2 to 12 ug/mL per 100 nM antisensecompound.

Cells are treated with antisense oligonucleotides by routine methodswell known to those skilled in the art. Cells are typically harvested16-24 hours after antisense oligonucleotide treatment, at which time RNAor protein levels of target nucleic acids are measured by methods knownin the art and described herein. In general, when treatments areperformed in multiple replicates, the data are presented as the averageof the replicate treatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art. Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM.

In vivo Testing of Antisense Oligonucleotides

Antisense oligonucleotides are tested in animals to assess their abilityto inhibit expression of a target nucleic acid and produce phenotypicchanges. Testing may be performed in normal animals, or in experimentaldisease models. For administration to animals, antisenseoligonucleotides are formulated in a pharmaceutically acceptablediluent, such as phosphate-buffered saline. Administration includesparenteral routes of administration, such as intraperitoneal,intravenous, and subcutaneous, and further includes intrathecal andintracerebroventricular routes of administration. Calculation ofantisense oligonucleotide dosage and dosing frequency is within theabilities of those skilled in the art, and depends upon factors such asroute of administration and animal body weight. Following a period oftreatment with antisense oligonucleotides, RNA is isolated from varioustissues and changes in target nucleic acid expression are measured.Changes in proteins encoded by target nucleic acids may also bemeasured. The types of phenotypic changes selected for monitoring aredependent upon the cellular pathway and disease with which the targetnucleic acid is associated.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.

Methods of RNA isolation are well known in the art. RNA is preparedusing methods well known in the art, for example, using the TRIZOL®Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of an SOD1 nucleic acid can beassayed in a variety of ways known in the art. For example, targetnucleic acid levels can be quantitated by, e.g., Northern blot analysis,competitive polymerase chain reaction (PCR), or quantitaive real-timePCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Quantitative real-time PCR can beconveniently accomplished using the commercially available ABI PRISM®7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitativereal-time PCR using the ABI PRISM® 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. Methods of quantitative real-time PCRare well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT, real-time-PCR reactions are carriedout by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as GAPDH, or by quantifying total RNA using RIBOGREEN®(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RIBOGREEN®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).Methods of RNA quantification by RIBOGREEN® are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000instrument (PE Applied Biosystems) is used to measure RIBOGREEN®fluorescence.

Probes and primers are designed to hybridize to an SOD1 target nucleicacid. Methods for designing real-time PCR probes and primers are wellknown in the art, and may include the use of software such as PRIMEREXPRESS® Software (Applied Biosystems, Foster City, Calif.). Primers andprobes useful for detection of human and rat SOD1 mRNA are described inU.S. application Ser. No. 10/672,866, published as US 2005/0019915,which is herein incorporated by reference in its entirety.

Analysis of Protein Levels

Protein levels of SOD1 can be evaluated or quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA),quantitative protein assays, protein activity assays (for example,caspase activity assays), immunohistochemistry, immunocytochemistry orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art. Antibodies useful for thedetection of human and rat SOD1 are well known in the art.

EXAMPLE 1 Inhibition of SOD1 mRNA in Rat Brain FollowingIntracerebroventricular Administration

In order to inhibit the gene expression in the central nervous system,antisense oligonucleotides must be delivered directly to thecerebrospinal fluid by, for example, intracerebroventricular (ICV)administration. To evaluate antisense inhibition of SOD1 in the brainsof normal rats, SOD1 mRNA levels were measured in rat brain followingICV administration. SOD1 mRNA levels were measured in both rat spinalcord and rat brain following ICV administration of ISIS 146192, anantisense oligonucleotide targeted to SOD1. Administration was performeddaily at either 33 μg/day or 50 μg/day for 14 days. ICV administrationof ISIS 146192 significantly reduced SOD1 mRNA levels in the spinal cordand right temporal parietal section of the brain. Thus, antisenseoligonucleotides that are delivered to the cerebrospinal fluid via ICVadministration are able to inhibit the expression of SOD1 in centralnervous system tissues that are affected in ALS. Accordingly, anembodiment of the present invention is the delivery of antisenseoligonucleotides to the cerebrospinal fluid by way of ICVadministration.

EXAMPLE 2 Antisense Inhibition of SOD1 in Human Fibroblasts

The A4V mutation of SOD1 accounts for 50% of SOD1-mediated familial ALSin the United States. Antisense oligonucleotides targeting SOD1 weretested for their ability to inhibit SOD1 expression in fibroblastsisolated from an individual harboring the A4V mutation. ISIS 333611,ISIS 333624 (complementary to nucleotides 440 to 459 of SEQ ID NO: 1),and ISIS 333636 (complementary to nucleobases 452 to 471 of SEQ IDNO: 1) inhibited SOD1 expression in a dose dependent manner when testedat doses of 3, 10, 30, 100, or 300 nM. SOD2 mRNA levels were notaffected.

EXAMPLE 3 Slowed Disease Progression in a Rat Model of Familial ALS

Several lines of transgenic mice and rats have been generated andextensively studied as experimental models of familial ALS. For example,transgenic mice have been engineered to express the human G85R SOD1variant. Transgenic rats expressing the human SOD1 G93A variant developsymptoms similar to ALS and do not survive beyond three to five monthsafter birth. As such, these transgenic rats are useful for the testingof antisense oligonucleotides targeted to SOD1. The presence of thehuman G93A SOD1 variant causes human SOD1 mRNA to accumulate to levelsapproximately 5 to 10 times that of endogenous wild-type rat SOD1.

Antisense oligonucleotide was infused into the right lateral ventricleof 65 day old rats expressing the human G93A SOD1 variant at a dose of100 ug/day for 28 days, using Alzet minipumps. Following the treatmentperiod, RNA was isolated from different regions of the brain, and SOD1mRNA levels were measured by real-time PCR. Despite the high level ofhuman SOD1 mRNA, ISIS 146145, ISIS 333611, ISIS 333624, and ISIS 333636were effective at reducing human SOD1 mRNA in different regions of thebrain. For example, ISIS 333611 effectively reduced human SOD1 mRNAlevels in the right cortex, cervical spinal cord, thoracic spinal cord,and lumbar spinal cord by approximately 69%, 45%, 50%, and 42%,respectively. Human SOD1 protein levels were reduced following treatmentwith ISIS 333611 by approximately 40% and 35%, respectively, in theright cortex and cervical spinal cord. Reduction of SOD1 protein wasgreater at one month than at two weeks, reflecting the known longhalf-life of SOD1 protein.

An additional study was performed to test the effects of antisenseinhibition of SOD1 on ALS disease onset in rats expressing the humanG93A SOD1 variant. Animals were treated by ICV infusion of 100 ug/day ofISIS 333611 (n=12) for a period of 28 days. Saline-treated (n=11)animals and control oligonucleotide-treated (n=8) animals served ascontrols. The timing of disease onset, at approximately 95 days of age,was similar in each group. While antisense inhibition of SOD1 did notslow early disease onset, infusion of ISIS 333611 slowed diseaseprogression, extending survival from 122±8 days to 132±7 days. Infusionof the control oligonucleotide had no effect on disease progression.

An embodiment of the present invention is a method of the slowing ofdisease progression in an individual suffering from familial ALS bydelivering to the cerebrospinal fluid an antisense oligonucleotidetargeted to SOD1. In other embodiments, the method further comprisesextending the survival of an individual suffering from familial ALS. Inpreferred embodiments, the antisense oligonucleotide is ISIS 333611.

EXAMPLE 4 Distribution of Antisense Oligonucleotides in Primate Tissues

To assess the distribution of antisense oligonucleotides followingdelivery to the cerebrospinal fluid, ISIS 13920 (an antisenseoligonucleotide having a gapped motif) was infusedintracerebroventricularly or intrathecally into non-human primates at adose of 1 mg/day for 14 days. A monoclonal antibody that recognizesoligonucleotides allowed for the immunohistochemical detection ofantisense oligonucleotide. Following ICV infusion, ISIS 13920distributed broadly throughout the central nervous system, with thehighest concentrations found in the cortex and the lowest concentrationsfound in the hypothalamus. Following IT infusion, antisenseoligonucleotides was broadly distributed throughout the central nervoussystem, with the highest concentrations of oligonucleotides found in thetissue adjacent to the site of infusion, the lumbar cord.

Accordingly, an embodiment of the present invention is the delivery ofantisense oligonucleotide targeted to SOD1 to the central nervoussystem, as well as the cerebrospinal fluid, throughintracerebroventricular or intrathecal infusion.

EXAMPLE 5 Administration of ISIS 333611 to Individuals Suffering fromFamilial ALS

The present invention provides methods of slowing disease progression inan individual suffering from familial ALS. Such methods comprise theadministration to the cerebrospinal fluid of the individual apharmaceutical composition comprising ISIS 333611. Delivery of thepharmaceutical composition to the cerebrospinal fluid allows for contactof the antisense oligonucleotide with the cells of central nervoussystem tissues, including tissues affected by ALS.

Individuals suffering from familial ALS receive a diagnosis of familialALS from a physician. The physician's assessment includes the ElEscorial criteria, genetic testing to verify the presence of a mutationin the SOD1 gene, and a neurological examination.

A Medtronic SyncroMed® II pump is used to deliver a pharmaceuticalcomposition comprising ISIS 333611 to the cerebrospinal fluid of anindividual suffering from familial ALS. The pump is surgically implantedper the procedures outlined by the manufacturer. Drug is retained in thereservoir of the pump, and is pumped at a programmed dose into acatheter that is surgically intrathecally implanted.

The reservoir is loaded with a pharmaceutical composition comprisingISIS 333611 in phosphate-buffered saline. The pharmaceutical compositionis administered at an amount that yields an infusion of 8 mg to 12 mg ofISIS 333611 into the cerebrospinal fluid. In preferred embodiments, theamount of ISIS 333611 infused is 10 mg. Administration is for a periodof at least 28 days.

Disease progression is measured by methods routine in the art anddescribed herein, for example, using the ALSFSR-R, and measurements ofFEV₁, FVC, and muscle strength. These methods are used by a physician toassess disease state at initiation of treatment, and this assessmentserves as a baseline for disease state. Subsequent assessments areperformed at regular intervals during the pharmaceutical compositiondelivery period; these intervals are determined by the physician.Administration of a pharmaceutical composition comprising ISIS 333611 tothe CSF of individuals suffering from familial ALS slows the progressionof ALS. In one embodiment, the ALSFSR-R score is not reduced relative tothe baseline ALSFSR-R score. In another embodiment, FEV₁ is not reducedrelative to baseline values. In an additional embodiment, FVC is notreduced relative to baseline values. In a further embodiment, musclestrength, such as hand grip strength, is not reduced relative tobaseline values.

1. A method of treating amyotrophic lateral sclerosis (ALS) comprisingadministering to the cerebrospinal fluid of a subject in need thereof atherapeutically or prophylactically effective amount of a compositioncomprising: a modified oligonucleotide consisting of 12 to 30 linkednucleosides having a nucleobase sequence comprising an 8-nucleobaseportion of SEQ ID NO: 3, wherein the modified oligonucleotide isspecifically hybridizable with SEQ ID NO: 1, and a pharmaceuticallyacceptable diluent or carrier.
 2. The method of claim 1, whereinadministering is intrathecal administration.
 3. The method of claim 1,wherein administering is intraventricular administration.
 4. The methodof claim 1, wherein at least one intenucleoside linkage is aphosphorothioate internucleoside linkage.
 5. The method of claim 1,wherein at least one nucleoside comprises a modified sugar.
 6. Themethod of claim 5, wherein the modified sugar is a 2′-O-methoxyethyl. 7.The method of claim 1, wherein at least one nucleoside comprises amodified nucleobase.
 8. The method of claim 7, wherein the modifiednucleobase is a 5-methylcytosine.
 9. The method of claim 1, wherein themodified oligonucleotide comprises: a gap segment consisting of linkeddeoxynucleosides; a 5′ wing segment consisting of linked nucleosides; a3′ wing segment consisting of linked nucleosides; wherein the gapsegment is position between the 5′ wing segment and the 3′ wing segmentand wherein each nucleoside of each wing segment comprises a modifiedsugar.
 10. The method of claim 9, wherein the modified oligonucleotidecomprises: a gap segment consisting often linked deoxynucleosides; a 5′wing segment consisting of five linked nucleosides; a 3′ wing segmentconsisting of five linked nucleosides; wherein the gap segment isposition between the 5′ wing segment and the 3′ wing segment, whereineach nucleoside of each wing segment comprises a 2′-O-methoxyethylsugar; wherein each cytosine is a 5′ methylcytosine, and wherein eachinternucleoside linkage is a phosphorothioate linkage.
 11. A method oftreating amyotrophic lateral sclerosis (ALS) comprising administering tothe cerebrospinal fluid of a subject in need thereof a therapeuticallyor prophylactically effective amount of a composition comprising: amodified oligonucleotide consisting of 12 to 30 linked nucleosideshaving a nucleobase sequence comprising an 8-nucleobase portion of SEQID NO: 3, wherein the modified oligonucleotide is 100% complementary toSEQ ID NO: 1, and a pharmaceutically acceptable diluent or carrier. 12.The method of claim 11, wherein administering is intrathecaladministration.
 13. The method of claim 11, wherein administering isintraventricular administration.
 14. The method of claim 11, wherein atleast one intenucleoside linkage is a phosphorothioate intenucleosidelinkage.
 15. The method of claim 11, wherein at least one nucleosidecomprises a modified sugar.
 16. The method of claim 15, wherein themodified sugar is a 2′-O-methoxyethyl.
 17. The method of claim 11,wherein at least one nucleoside comprises a modified nucleobase.
 18. Themethod of claim 17, wherein the modified nucleobase is a5-methylcytosine.
 19. The method of claim 11, wherein the modifiedoligonucleotide comprises: a gap segment consisting of linkeddeoxynucleosides; a 5′ wing segment consisting of linked nucleosides; a3′ wing segment consisting of linked nucleosides; wherein the gapsegment is position between the 5′ wing segment and the 3′ wing segmentand wherein each nucleoside of each wing segment comprises a modifiedsugar.
 20. The method of claim 19, wherein the modified oligonucleotidecomprises: a gap segment consisting often linked deoxynucleosides; a 5′wing segment consisting of five linked nucleosides; a 3′ wing segmentconsisting of five linked nucleosides; wherein the gap segment isposition between the 5′ wing segment and the 3′ wing segment, whereineach nucleoside of each wing segment comprises a 2′-O-methoxyethylsugar; wherein each cytosine is a 5′ methylcytosine, and wherein eachinternucleoside linkage is a phosphorothioate linkage.